Vitamin D binding protein (DBP) is an abundant serum protein belonging to the multigene family that includes albumin, alpha- fetoprotein, and alpha-albumin/afamin. Multiple functions have been attributed to DBP. It binds vitamin D sterols facilitating their serum transport; it sequesters G-actin released to the serum after cellular damage and along with plasma gelsolin constitutes the hypothesized "plasma actin-scavenger" non-immune host defense system; and it activates macrophages and participates in leukocyte chemotaxis in in vitro models. We recently generated a DBP-/- (null) mouse model and characterized its vitamin D metabolism. This mouse line will constitute the model system to determine DBP's in vivo roles in immune function. Aim 1 will addresses the hypothesis the DBP functions as a macrophage-activating factor in vivo. Macrophages from the peritoneal, pulmonary, bone marrow, and bone (osteoclasts) compartments of DBP-/- and DBP+/+ mice will be compared and characterized for biological functions that define the activated state. The functions attributable to DBP will be carefully dissected from those attributable to its vitamin D sterol delivery function because 1,25(OH)2D is itself a potent macrophage differentiating factor. This will be done by analyzing the actions of a DBP transgene with a discrete mutation in its vitamin D binding domain. In Aim 2, DBP's role in host defense will be studied using an endotoxin shock model and by studying the response of DBP-/- mice to infections with a set of macrophage intracellular pathogens. In Aim 3, the DBP chemotaxis domain will be mapped functionally by analysis of site-directed mutations in vivo, DBP's ability to signal through Ca2+ mobilization will be explored, and the role of megalin in macrophage uptake of DBP will be determined. In Aim 4, the actin-scavenger hypothesis will be tested by quantifying pulmonary damage in DBP-/-, GSN-/-, and DBP-/-GSN-/- mice after iv G-actin overload. These studies, utilizing a combination of in vitro, cell-based, and in vivo models, will document novel, non-redundant roles for DBP in inflammation and in immune as well as non-immune host defense.